Class II LitR serves as an effector of “short” LOV-type blue-light photoreceptor in Pseudomonas mendocina

PmlR2, a class II LitR/CarH family transcriptional regulator, and PmSB-LOV, a “short” LOV-type blue light photoreceptor, are adjacently encoded in Pseudomonas mendocina NBRC 14162. An effector protein for the “short” LOV-type photoreceptor in Pseudomonas has not yet been identified. Here, we show that PmlR2 is an effector protein of PmSB-LOV. Transcriptional analyses revealed that the expression of genes located near pmlR2 and its homolog gene, pmlR1, was induced in response to illumination. In vitro DNA–protein binding analyses showed that recombinant PmlR2 directly binds to the promoter region of light-inducible genes. Furthermore PmSB-LOV exhibited a typical LOV-type light-induced spectral change. Gel-filtration chromatography demonstrated that the illuminated PmSB-LOV was directly associated with PmlR2, whereas non-illuminated proteins did not interact. The inhibition of PmlR2 function following PmSB-LOV binding was verified by surface plasmon resonance: the DNA-binding ability of PmlR2 was specifically inhibited in the presence of blue light-illuminated-PmSB-LOV. An In vitro transcription assay showed a dose-dependent reduction in PmlR2 repressor activity in the presence of illuminated PmSB-LOV. Overall, evidence suggests that the DNA-binding activity of PmlR2 is inhibited by its direct association with blue light-activated PmSB-LOV, enabling transcription of light-inducible promoters by RNA polymerase.


Results
pmlR and pmSB-LOV genes of Pseudomonas mendocina NBRC 14162. P. mendocina NBRC 14162 is a gram-negative bacterium isolated by soil enriched with ethanol as a carbon source 22 . Figure 1 shows a schematic representation of the genomic organization of two pplR homologs (designated pmlR1 and pmlR2) and a homolog of ppSB-LOV (designated pmSB-LOV) in P. mendocina NBRC14162.
The pmlR2 gene encodes a protein of 321 amino acids (calculated molecular weight: 35.82 kDa) and shares 56%, 39%, and 34% identity with PplR3, PplR2, and PplR1 of P. putida, respectively (Fig. S1A). In contrast, PmlR1 (318 aa, 35.56 kDa) shares 45%, 36%, and 28% amino acid identity with PplR1, PplR3, and PplR2, respectively. PmlR2 shares a low identity (26.4%) with PmlR1 across the entire sequence, but its N-terminal MerR-type HTH DNA-binding domain shares 70% similarity. This high similarity in the HTH domain was also observed for PplR proteins (Fig. S1A). A Pfam search showed that the internal regions of PmlR1 and PmlR2, residues Arg 106 to Arg 179 (PmlR1) and Trp 113 to Leu 161 (PmlR2), share similarity with a B 12 -binding_2 domain (PF02607) with E-values of 1.1e-05 and 3.5e-07, respectively. The C-termini of both PmlR proteins shared no similarity with known functional protein domains. Generally, two domains, the B 12 -binding domain and the B 12 -binding_2 domain, are required for association with B 12 , suggesting that PmlRs have no ability to form a complex with B 12 .
PmSB-LOV consists of 148 amino acids (16.68 kDa), including a typical "short" LOV protein lacking an effector domain. PmSB-LOV shares 100% identity with MDS_3586 of P. mendocina NK-01 (Fig. S1B), and end-to-end similarity with PpSB2-LOV (71%) and PpSB1-LOV (69%) of P. putida KT2440. A conserved cysteine residue required for the thiol-adduct formation with FMN was also present at the position 53 (designated as Cys 53 ) of the PmSB-LOV protein. The conserved N-terminal N-cap and C-terminal Jα-helix found in other "short" LOVs was also found in PmSB-LOV (Fig. S1B). Light-inducible transcription in P. mendocina NBRC 14162. We performed RNA-seq analysis to preliminary screen genes whose expression is changed in response to blue light. Total RNA was purified from cells cultured in liquid medium under dark and blue light conditions at an intensity of 30.5 µmol s −1 m −2 . Lightinduced genes more than two-fold are summarized in Table S1. The identified light-inducible genes were common to P. putida KT2440, as previously reported by us 9 .
To confirm the results of RNA-seq analysis, we performed quantitative RT-PCR (qRT-PCR). The transcription of pmlR1, phrB, and ufaM was much higher under light conditions ( Table 1). Transcription of pmlR2 and pmSB-LOV was constitutive under dark and light conditions. Thus, many of the genes located in the flanking region of pmlR1 were strongly transcribed in a photo-dependent manner, and those of pmlR2 were slightly upregulated in light conditions. phrB and ufaM in P. putida KT2440 are similarly regulated by PplRs, suggesting that these genes are regulated by PmlR1 and PmlR2 in this bacterium.
We predicted the transcriptional units based on the results of RNA-seq and qRT-PCR analyses. Nine genes (PME6650 to PME6610), two genes (PME7070 and cryB), and three genes (folM, folE, and folX) likely comprise a polycistron, transcribed by the promoter preceding the first gene, as their transcriptional levels detected by Gene organization of the pmlR1 and PmlR2 genomic regions. P6650 is a light-inducible promoter analyzed in this study. The homologous genes with known functions include phrB, a deoxyribodipyrimidine photo-lyase; PME6630, a short-chain dehydrogenase; PME6625, a FAD-dependent oxidoreductase; ufaM, a furan-containing fatty acid synthase; cryB, a cryptochrome/photolyase family protein; folX, a dihydroneopterin triphosphate 2′-epimerase; folE, a GTP cyclohydrolase I; and folM, a dihydromonapterin reductase. Other ORFs encode proteins without annotated/known function. www.nature.com/scientificreports/ RNA-seq analysis showed a similar fold change in response to illumination (Table S1). The putative translational start codon for each downstream coding sequence from the first gene of the operon overlapped or was close with the stop codon of the preceding coding sequence. It is important to understand the in vivo function of the pmlR and pmSB-LOV genes in P. mendocina NBRC 14162. However, we were unable to establish a transformation method by conjugative transfer or electroporation using several types of vectors, including a broad-host-range plasmid (pBHR1) and genome-integrative transposon (pUTminiTn5). Thus, we mainly focused on the in vitro analysis of PmlR2 and PmSB-LOV.
Promoter structure preceding the light-inducible PME6650 gene. To assign the transcriptional start site of the PME6650 promoter (designated P6650) of P. mendocina NBRC 14162 and PP0468 (designated P468) of P. putida KT2440 used for the control promoter in vitro transcriptional runoff assay as described below, we performed 5′-RACE and CAGE experiments. The transcriptional start sites of P6650 and P468 were located 48 bp and 51 bp upstream of the ATG translation initiation codon ( Fig. 2A). The −35 and −10 regions of P6650 (TTG ACT N16 TAT GAT ) were highly similar to the consensus sequence of the Escherichia coli σ 70 -dependent promoter (TTG ACA N16-18 TAT AAT ) 26 . Based on the position of the transcriptional start site, we annotated a different translational initiation codon for PME6650 in P. mendocina NBRC 14162 than that in P. mendocina NK-01.
We first evaluated the DNA-binding activity of the recombinant PmlR2 protein using a gel-shift assay. PmlR2 was expressed as a Glutathione-S-Transferase (GST)-fusion protein in E. coli and purified using GST affinity chromatography. The GST-tag was removed using a sequence-specific protease (See Materials and Methods), and the tag-free PmlR2 recombinant protein was used for the gel-shift assay and other experiments. PmlR2 bound to the promoter region of PME6650 and folM but did not bind to the rpoD promoter region at low protein concentrations (Fig. S2). Among these promoter regions, the most stable band shift was observed when P6650 was used as the probe. Therefore, we used P6650 to study PmlR2 DNA-binding activity in this study.
DNase I footprint analysis was used to identify the PmlR2-binding sites on the sense and antisense strands of P6650 ( Fig. 2A and B). Single broad regions in the sense strand (+ 32 to − 45) and antisense strand (+ 37 to − 54) were protected from DNase I treatment, indicating that P6650 contains multiple PmlR2-binding sites (Fig. 2B). The supporting data shown in Fig. S3 was used to analyze the result of the sense strand in Fig. 2B, as the specific DNA-binding activity of PmlR2 with the sense strand was low compared to that of the antisense strand. We compared the nucleotide sequences of the protected regions with the consensus of PplR1 9 . As shown in Fig. 2A and C, the nucleotide sequences 5′-TGT ACA -N 12 -TGT ACA -3' , designated as Site 1, and 5′-TGT ACA -N 12 -TGT ACA -3' , designated as Site 2, shared high similarity with the consensus sequences (5′-T(G/A)TACA-N 12 -TGTA(C/T) A-3') of PplR1 9 . Site 1 overlaps with the − 35 region of P6650, Site 2 is located between the transcriptional start site and the translational initiation codon of PME6650.

Light-dependent change in the absorbance spectrum of PmSB-LOV recombinant protein. The
LOV protein generally shows light-dependent absorption changes (called photocycles) in the UV-visible region (300-600 nm) due to the formation of a thiol adduct between the conserved Cys residue of LOV and FMN upon illumination. The LOV domain absorption ability is restored to the dark state in the absence of light (dark state recovery). To investigate the changes in absorption, we purified wild-type PmSB-LOV and PmSB-LOV C53A recombinant proteins from an E. coli expression host (See Materials and Methods). Both purified proteins were yellow, consistent with their association with flavin derivatives, characteristic of LOV proteins. HPLC analyses showed that both types of PmSB-LOV proteins were predominately associated with FMN and a smaller amount of FAD chromophore (Fig. S4). This result indicates two things: (i) FMN is a major chromophore of PmSB-LOV, and (ii) the Cys 53 residue is not involved in the association with flavins.
We then measured the absorbance spectrum (300-600 nm) of PmSB-LOV in dark and light conditions at 25 °C. The absorption spectrum of dark-incubated PmSB-LOV contained multiple peaks, similar to a typical LOV profile. The PmSB-LOV spectrum changed upon exposure to 450 nm blue light at 100 μmol s −1 m −2 for 10 s (Fig. 3A), whereas exposure to light at 365, 530, or 660 nm had no effect. Thus, PmSB-LOV exhibited typical absorbance spectrum behavior LOV proteins.
We also analyzed the spectra of PmSB-LOV C53A to assess the involvement of the conserved Cys 53 residue in the change in absorbance. Unlike the wild-type protein, PmSB-LOV C53A exhibited similar absorption spectra under both dark and light conditions (Fig. 3A), suggesting that the Cys 53 residue is required for the formation of the thiol-adduct with FMN, similar to other LOV proteins.
Next, the absorbance was monitored at 450 nm for 1 h to trace the dark state recovery. Full recovery to the dark state was observed after 56 min (Fig. 3B). This indicated that PmSB-LOV is a relatively slow-reverting LOV protein.

Protein-protein interaction between PmlR2 and PmSB-LOV in heterologous E. coli.
To obtain preliminary information on the interaction between PmlR2 and PmSB-LOV in vivo, we performed a two-hybrid analysis using E. coli as a heterologous host. In each construct, PmlR2 and PmSB-LOV were fused at the C-terminal domain of each split cAMP synthase domain (T18 and T25). As shown in Fig. 4, E. coli transformants carrying pmlR2 and ppSB-LOV exhibited higher β-galactosidase activity than the negative (empty/empty) and positive (zip/zip) control strain. The β-galactosidase activities generated by the interaction were observed under dark and light conditions. We performed the same analysis with E. coli transformants carrying pmlR2 and ppSB-LOV, in which both genes were fused with various pairs of cAMP synthase domains (Fig. S5). We assume that chimeric construction via C-terminal or N-terminal fusion with the cAMP synthase domain prevents lightdependent protein-protein interaction.

Light-dependent complex formation composed of PmlR2 and PmSB-LOV.
We performed a gelshift assay in the presence of PmSB-LOV to examine the association between PmlR2 and PmSB-LOV. When P6650 was used as the probe, the addition of non-illuminated PmSB-LOV caused a partial super shift (Fig. 5).
On the other hands, the addition of white light-treated PmSB-LOV caused a clear super shift, and a partial dose-dependent super-shift was observed (Fig. 5). This strongly suggests that illuminated PmSB-LOV directly interacts with PmlR2, while non-illuminated PmSB-LOV has the low activity.

Light-dependent protein-protein interaction between PmlR2 and PmSB-LOV.
To confirm the light-induced interaction between PmSB-LOV and PmlR2 observed in the gel-shift assay, the gel-filtration chromatography analysis was performed. Single proteins or protein mixtures incubated under dark or light conditions were separated using gel-filtration chromatography. When each protein was separately loaded onto the column, PmlR2 and PmSB-LOV eluted at approximately 226 kDa and 51. 8   www.nature.com/scientificreports/ state ( Fig. 6A and B), suggesting that PmlR2 and PmSB-LOV exist in a hexamer and trimer, respectively, in solution.
When PmlR2 and PmSB-LOV were co-incubated in the dark, they were eluted as two major peaks (Fig. 6D). Each elution volume was almost identical to those of PmlR2 and PmSB-LOV. SDS-PAGE and silver staining confirmed that the former and latter peaks contained PmlR2 and PmSB-LOV, respectively. Thus, incubation in dark did not affect the interaction between PmSB-LOV and PmlR2. In contrast, illuminated mixtures of PmlR2   www.nature.com/scientificreports/ and PmSB-LOV generated a new peak (Fig. 6D) that eluted at a higher volume. The new peak contained PmlR2 and PmSB-LOV, whereas the latter two peaks were eluted in almost same fraction as PmlR2 and PmSB-LOV, respectively. Therefore, PmlR2 and PmSB-LOV interacted in response to illumination. We examined PmSB-LOV C53A using the same method. As shown in Fig. 6C, PmSB-LOV C53A did not associate with PmlR2 under dark or light conditions. Our results indicate that PmSB-LOV directly associates with PmlR2 in response to illumination, and Cys 53 may be involved in this light-dependent association.

N-terminal domain of PmlR2 interacts with PmSB-LOV.
To determine the interaction domain between PmlR2 and PmSB-LOV, two truncated proteins, (i) PmlR2-N protein containing only the HTH DNAbinding domain and (ii) PmlR2-C protein containing only the C-terminal domain, were subjected to gel-filtration chromatography analysis. The calculated molecular weights of the PmlR2-N and PmlR2-C recombinant proteins with the GPLGS sequence were 12.8 kDa and 25.9 kDa, respectively. When each protein was separately loaded onto the column, PmlR2-N and PmlR2-C eluted at approximately 22 kDa and > 440 kDa, respectively, regardless of the illumination state (Figs. S6 and S7), suggesting that PmlR2-N and PmlR2-C likely exist as dimers and oligomers, respectively. When PmlR2-N and PmSB-LOV were co-incubated in the dark, they eluted as two major peaks. Each elution volume was almost identical to those of PmlR2-N and PmSB-LOV (Fig. S6). In contrast, the illuminated mixtures of PmlR2-N and PmSB-LOV generated a new peak (Fig. S6), which eluted at a higher volume and contained both PmlR2 and PmSB-LOV. Therefore, PmlR2-N interacted with PmSB-LOV www.nature.com/scientificreports/ in response to illumination. We examined the interaction between PmlR2-C and PmSB-LOV using the same method. As shown in Fig. S7, PmlR2-C is not associated with PmSB-LOV under dark or light conditions. Our results indicate that the PmlR2-N domain directly interacts with PmSB-LOV in response to illumination and also suggests that the PmlR2-N domain prevents oligomerization of its C-terminal domain.

Light-dependent inhibition of the PmlR2 DNA-binding activity by PmSB-LOV. Our previous
genetic study on P. putida KT2440 suggested that PplR serves as a negative regulator, and its activity is controlled by PpSB-LOV. Therefore, we studied the function of PmSB-LOV by examining whether PmlR2 DNA-binding activity is inhibited in the presence of PmSB-LOV using surface plasmon resonance spectroscopy (BIAcore). The promoter region preceding PP_0468 was used as a negative control since PmlR2 did not bind to this region in gel-shift assays (data not shown). A biotin-conjugated oligonucleotide containing PmlR2-binding Site 2 of P6650 (− 9 to + 41, Fig. 2A) was fixed to the sensor chip SA. No PmlR2-binding response was observed when P468 was immobilized on the sensor chip (Fig. 7A). In contrast, a dose-dependent increase in response units was observed following the infusion of increasing amounts of PmlR2 over Site 2 bound to the immobilized chip (Fig. 7B), indicating that PmlR2 bound to Site 2.
We examined the effect of PmSB-LOV on the DNA-binding activity of PmlR2 (Fig. 7C). The PmlR2 DNAbinding activity was inhibited in the presence of illuminated PmSB-LOV. This inhibition was caused by blue-light illumination for 90 s; in contrast, dark incubation or illumination with 365 nm, green, or red light had no effect (Fig. 7D). We also investigated the dark state recovery of the PmSB-LOV after illumination with blue light. A large increase in response units was observed, representing PmlR2-binding during a long dark incubation period (Fig. 7E). Thus, dark incubation caused a loss of inhibitory function due to dark state recovery of PmSB-LOV.
We also examined the function of PmSB-LOV C53A (Fig. 7F), which did not inhibit the DNA-binding activity of PmlR2. We confirmed that illumination did not affect the DNA-binding affinity of PmlR2 and that PmSB-LOV has no DNA-binding activity (Fig. 7G). We also confirmed specific binding of PmlR2 to Site 2 (TGT ACA -N 12 -TGT ACA ). PmlR2 did not bind dsDNA containing a mutated Site 2 (AAG CTT -N 12 -AAG CTT ), in which both consensus TGT ACA sequences were replaced with AAG CTT (Fig. 7H). Our data suggest that the DNAbinding activity of PmlR2 is inhibited by blue-light activated PmSB-LOV, and Cys 53 is required for its inhibitory function. Moreover, dark incubation of PmSB-LOV eliminates the inhibitory function due to dark state recovery.

PmSB-LOV serves as an anti-repressor against PmlR2. We suggest that PmSB-LOV senses blue light
and associates with PmlR2 to inhibit its DNA-binding activity. To clarify whether PmSB-LOV directly inhibits PmlR2 function in vitro, we performed an in vitro transcriptional runoff assay using components required for mRNA synthesis and regulation. We used RNA polymerase holoenzyme containing an essential sigma factor σ 70 , a commercially available protein derived from E. coli, because the -10 and − 35 sequence of P6650 is similar to that of the E. coli σ 70 -dependent promoter. Pseudomonas σ 70 is insoluble and, thus, could not be used in this study.
The RNA polymerase holoenzyme generated a transcript of the expected length from the template containing P6650 or P468 promoters (Figs. 8A, S8), whereas the core enzyme of the E. coli RNA polymerase produced small amount of transcript. To examine whether PmlR2 serves as a repressor, we added PmlR2 to the transcription reaction. PmlR2 dose-dependently inhibited the mRNA synthesis from P6650. Inhibition was not observed when P468 was used as a template; thus, PmlR2 specifically inhibited the transcription from P6650.
We then assessed whether PmSB-LOV has a negative regulatory effect on PmlR2 activity in response to light. Illuminated or non-illuminated PmSB-LOV were added to the transcription reaction in the presence of PmlR2. The addition of illuminated PmSB-LOV restored transcription in a dose-dependent manner despite the presence of PmlR2 (Figs. 8B, S8). However, the presence of non-illuminated PmSB-LOV did not affect the transcriptional efficiency. No inhibition was observed in the presence of PmSB-LOV. Our results demonstrated that PmlR2 serves as a repressor and that PmSB-LOV serves as a light-dependent anti-repressor of PmlR2 activity.

Discussion
The biochemical properties of the bacterial "short" LOV proteins have been well studied, but their effector proteins have not been identified. Our study revealed that the MerR family transcriptional regulator PmlR2 is an effector protein of PmSB-LOV in P. mendocina NBRC14162. This is the first report on the identification of a protein partner for a "short" bacterial LOV protein. Furthermore, in the bacterial photo-response system, the molecular mechanism underlying the interaction between the MerR family regulator and the LOV is unknown. Therefore, the regulatory system identified in this study is a novel photo-regulatory mechanism. The conservation of PmlR2 and PmSB-LOV homologs in Pseudomonas spp. and closely related bacteria indicates that light-sensing and responses are required for survival in nature. Figure 9 illustrates the regulatory model based on the results of this study. Under dark conditions, PmlR2 associates with binding sites in the promoter region of PME6650, preventing access by RNA polymerase. Upon illumination with blue light, FMN-bound PmSB-LOV is converted to its activated form, which interacts directly with PmlR2. This protein-protein interaction abolishes the DNA-binding activity of PmlR2, thus permitting transcription from the light-inducible promoters by RNA polymerase. When the environment returns to dark conditions, PmSB-LOV becomes inactivated due to dark-state recovery, and free PmlR2 binds to the promoter region, inducing the OFF state.
The in vitro transcriptional runoff assay clearly demonstrated that the base function of PmlR2 is a repressor, which is fundamental to preventing the expression of light-inducible genes when they are not required, similar to the function of many LitR/CarH family members. PmlR2 bound to a consensus sequence similar to that of PplR1 (Fig. 2C)  www.nature.com/scientificreports/ PplR1. Consensus was also found 95 bp upstream of the translational initiation site (5′-TGT ACA -N 12 -GGT ATA -3') for the light-inducible gene, folM. Thus, PmlR2 may bind to these sequences to control their transcription. Comprehensive analysis such as chip-seq will permit a more thorough evaluation of PmlR2 binding sites in future studies. PmlR2 contains an internal B 12 binding domain and C-terminal domain of unknown function (Fig. S1A). Many previous studies have shown that the B 12 -dependent photosensor LitR of T. thermophilus has two C-terminal B 12 -binding domains (B 12 -binding_2 and B 12 -binding domain) that mediate AdoB 12 -binding 1,27 . The latter domain contains a motif essential for the association with B 12 . The internal domain of PmlR2 (residues Trp 113 to Leu 161 ) is similar to the B 12 binding_2 domain with an E-value of 1.1e-05, while the C-terminal domain of PmlR2   9 . We assume that the C-terminus serves as an oligomerization domain based on the results of gel-filtration analysis (Fig. S7). The PmSB-LOV was predominantly associated with FMN (Fig. S4) and exhibited typical LOV domain-type properties such as light-induced spectral changes and dark state recovery (Fig. 3A). The adduct state lifetime of PmSB-LOV was similar to that of PpSB1-LOV in P. putida KT2440 (109 ± 18 min for PpSB1-LOV, 0.5 ± 0.1 min for PpSB2-LOV). The dark state recovery of PmSB-LOV was also observed in the BIAcore analysis (Fig. 7E).
The conformation of the well-studied "short" LOV protein is largely affected by illumination: RsLOV of Rhodobacter sphaeroides shows light-induced subunit dissociation 28 . In contrast, PmSB-LOV and PpSB1-LOV [16][17][18] constitutively formed dimers in solution, regardless of the light conditions (Fig. 6). The light-state crystal structure of PpSB1-LOV suggests that the N-terminal N-cap and C-terminal Jα-helix located at the subunit interface stabilize the parallel homodimer via coiled-coil interactions. The N-cap and Jα-helix are strictly conserved The anti-repressor activity of PmSB-LOV. P6650 was used as a template. The RNA polymerase holoenzyme containing σ 70 (E σ 70 ) or its coreenzyme, PmlR2, and PmSB-LOV were added to the reaction mixture with 0.5 pmol template in the indicated amount, and the proteins were marked as presence (+) or absence (−). The generated transcripts for P468 and P6650 were analyzed by 6 M urea non-denaturing PAGE, followed by autoradiography. Two independent experiments in technical replicates were performed, and representative data were shown. The original and unprocessed versions of the full-length gels were included in Fig. S14 and S15.

Dark
Blue Light www.nature.com/scientificreports/ among "short" LOV proteins, including PmSB-LOV (Fig. S1B), suggesting that a light-induced conformational change, but not subunit dissociation or association, is necessary for activity switching in PmSB-LOV. The threedimensional structures of the protein in dark and light will reveal the molecular mechanism underlying the functional changes in response to illumination. The conserved Cys 53 residue was essential for light-inducible activation of PmSB-LOV, as it was required for the light-induced absorption spectrum change (Fig. 3A), the interaction with PmlR2 (Fig. 6), and PmlR2 inhibitory activity (Figs. 7, 8). In contrast, the Cys 53 residue was not involved in the association with FMN (Figs. 3A and S4) or dimerization (Fig. 6). These results indicate that the formation of a covalent bond between Cys 53 and position 4a of the flavin chromophore is the foundation of the light-induced functional change in PmSB-LOV, generating the active form of PmSB-LOV upon light excitation. Thus, the Cys 53 residue is crucial to light-sensing by PmSB-LOV.
Our analysis revealed that the N-terminal HTH domain of PmlR2 interacts with PmSB-LOV (Fig. S6). The short N-terminal extension and Jα-helix of the LOV domain of the EL222 protein in the marine bacterium Erythrobacter litoralis interact with an HTH domain in its C-terminus 29 . This suggests that the Jα-helix of PmSB-LOV (Fig. S1B) interacts with the HTH domain of PmlR2 (Fig. S1A). Further analysis is required to characterize the molecular mechanism underlying the interaction.
Our results provide the first evidence of interaction between a "short" LOV protein and partner protein, since gel-shift assay (Fig. 5) and gel-filtration chromatography (Fig. 6) demonstrated the light-induced protein-protein interaction between PmlR2 and PmSB-LOV. Furthermore, we report that an LOV functions as an anti-repressor, since BIAcore analysis demonstrated that illuminated PmSB-LOV inhibits the DNA-binding activity of PmlR2 (Fig. 7), and the in vitro transcriptional runoff assay demonstrated that blue-light-activated PmSB-LOV serves as an anti-repressor of PmlR2 (Fig. 8). We also observed dark recovery with BIAcore analysis and found that the time required for dark recovery was similar to the absorbance spectra after 60 min. Our study revealed that PmSB-LOV directly associates with PmlR2 in a light-inducible manner; however, the intramolecular mechanism and structure of PmSB-LOV are unknown. Elucidation of the three-dimensional structures of the dark and light states may shed light on the precise light-response mechanism.
The gel-shift assay and BIAcore analysis showed different results in the complex formation of PmlR2, PmSB-LOV, and probe DNA under light conditions. The gel-shift assay showed the formation of a stable PmlR2/PmSB-LOV complex bound to P6655 (right panel of Fig. 5), while BIAcore analysis showed inhibition of PmlR2 DNAbinding activity by PmSB-LOV (Fig. 7C). In both experiments, the PmlR2/PmSB-LOV complex was formed under light conditions in advance, and followed by analysis of the interaction with the probe. In the gel-shift assay, gels were continuously illuminated with light from an external source for approximately 120 min of electrophoresis. In contrast, in BIAcore analysis, the time required for the protein complex to come into contact with the probe was 1-2 min. Therefore, the inconsistency of the results between the two experiments may be due to the different experimental conditions. We favor the result of the BIAcore analysis as it requires less analysis time.
This study also provides insights into the role of MerR family regulator. Two types of MerR family proteins involved in the light-response system of bacteria have been identified: (i) the light-sensitive AdoB 12 -based LitR/ CarH family and (ii) E. coli BluR controlled by the blue light receptor BluF 30 . Our study reveals a new type, (iii) PmlR2 regulated by a "short" LOV protein. The wide distribution of LitR/CarH family homologs leads us to assume the diversity of an unidentified photo-response mechanisms in bacteria.

Materials and methods
Bacterial strains, plasmids, and culture media. Pseudomonas mendocina NBRC 14162 22 was obtained from the Biological Resource Center, NITE (NBRC), Kazusa, Japan. Escherichia coli (E. coli) strains HST08 and Rosetta2(DE3)pLysS (Takara Bio Inc., Shiga, Japan) were used as hosts for DNA manipulation and protein expression, respectively. pUC118 (Takara Bio) was used for general DNA manipulation. pMD19 (Takara Bio) was used for TA-cloning of PCR-generated DNA fragments. pGEX-6P-2 (GE Healthcare UK Ltd, Buckinghamshire, England) was used to overexpress PmlR2 and PmSB-LOV in E. coli. Enzymes for DNA manipulation were purchased from Takara Bio. Culture conditions and genetic manipulations in E. coli are described by Maniatis et al. 31 E. coli and P. mendocina were grown at 28 °C or 30 °C in Luria-Bertani (LB) medium 31 ; 1.5% agar (Kokusan, Tokyo, Japan) was added to the solid media. Antibiotics were added to the following final concentrations: ampicillin and kanamycin at 50 μg/mL; chloramphenicol at 15 μg/mL.
Total RNA preparation and quantitative RT-PCR. Total RNA was purified from P. mendocina with an RNeasy Protect Bacteria kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. P. mendocina was cultured at 28 °C in liquid LB under blue light (30.5 µmol s −1 m −2 ) and dark conditions. cDNA was synthesized with SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) as described by the manufacturer. The amount of the synthesized cDNA was quantified with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) according to the manufacturer's instructions and our previous study 8 . The oligonucleotide primers were summarized in Table S2. Relative gene expression was quantified using the signals of dnaA (an essential chromosomal www.nature.com/scientificreports/ replication initiator gene) as an internal reference and the relative quantitative 2 −ΔΔCt method 32 . All reactions were performed in triplicate.

RNA-seq analysis.
For sequencing library preparation, 1 µg of total RNA was prepared using the following methods. Ribosomal RNA was removed using the RiboZero bacteria kit (Illumina, San Diego, CA, USA), according to the manufacturer's protocol. Sequencing libraries were prepared by NEBNext mRNA Library Prep Kit from Illumina (New England Biolabs, Ipswich, MA, USA) with the following modifications. A random hexamer primer was used for reverse transcription. After second-strand synthesis, the double-stranded cDNA was fragmented to an average length of 300 bp using a Covaris S2 sonication system (Covaris, Woburn, CA, USA). One hundred cycles of paired-end sequencing were carried out using HiSeq2500 system according to the manufacturer's specifications (Illumina). After sequencing reactions were complete, the Illumina analysis pipeline (CASAVA 1.8.0) was used to process the raw sequencing data. The RNA-Seq reads were trimmed using the CLC Genomics Workbench ver. 9.0.1 (Qiagen, Venlo, Netherlands) with the following parameters; Quality score: 0.05; Removing terminal 15 nucleotides from 5' end and 2 nucleotides from 3' end; Trimming read through adaptor: yes; Removing truncated reads less than 30 nucleotides in length. Trimmed reads were mapped to all genes in P. mendocina NBRC 14162 (accession number: NZ_BBQC00000000.1) using the CLC Genomics Workbench ver. 9.0.1. with the following parameters; Length fraction: 0.7; Similarity fraction: 0.9; Maximum number of hits for a read: 1. The expression level of each gene was calculated by counting the mapped reads for each gene and were normalized by calculating RPKM values. Original sequence reads were deposited to DRA/ SRA database under the accession number (DRA014932). The biological replicate in the RNA-seq experiment was n = 1, because the purpose of RNA-seq in this study was for preliminary screening of light-inducible genes.
Determination of transcriptional start sites. Transcriptional start sites were determined by 5′-RACE (rapid amplification of cDNA ends) and CAGE experiments using the total RNA purified from light-irradiated cells. For 5′-RACE, sequencing of the 5′-ends of the PME6650 and PP0468 transcripts was carried out with the 5′-Full RACE Core Set (Takara Bio) according to the manufacturer's instructions. The used oligonucleotides, ORF4-RT, ORF4-A1, ORF4-A2, ORF4-S1, and ORF4-S2 for PME6650, and PP468-RT, PP468-A1, PP468-A2, PP468-S1, and PP468-S2 for PP0468 were shown in Table S3. The amplified DNA fragments were cloned into the T-vector, pMD19 and the cloned inserts were sequenced by Eurofins Genomics. For the CAGE experiment, CAGE library preparation, sequencing, mapping, and gene expression and motif discovery analysis were performed using DNAFORM (Yokohama, Kanagawa, Japan). In brief, RNA quality was assessed using a Bioanalyzer (Agilent) to ensure that RNA integrity number (RIN) is over 7.0, and the A260/280 and 260/230 ratios are over 1.7. First strand cDNAs were transcribed to the 5' end of capped RNAs, attached to CAGE "bar code" tags and the sequenced CAGE tags were mapped to the P. mendocina genomes using BWA software (v0.5.9) after discarding ribosomal RNAs. For tag clustering, CAGE tag count data were clustered using CAGEr 33 Table S3). The expression vector for PmSB-LOV C53A , in which Cysteine 53 (Cys 53 ) was replaced with alanine, was generated using twostage PCR. The upstream and downstream regions of the mutation were amplified using the primers LOVex-F/ C53A-MR and C53A-MF/LOVex-R, respectively. The first-round PCR amplicons served as a template for the second-round PCR with the primers LOVex-F/LOVex-R. The 3' ends of the upstream first-round PCR products were complementary to the downstream region, thus generating a fusion construct in the second-round PCR. The amplified DNA fragments were digested with BamHI and EcoRI, and then cloned into the same site of pGEX-6P-2 (GE Healthcare) to generate pGEX-PmlR2 (PmlR2), pGEX-PmlR2-N(PmlR2-N), pGEX-PmlR2-C(PmlR2-C), pGEX-LOV (PmSB-LOV), and pGEX-C53A (PmSB-LOV C53A ). The insertions were verified by sequencing using an ABI3100 Genetic Analyzer. The recombinant gene encodes a protein with N-terminal GST under the control of an isopropyl β-D-thiogalactoside (IPTG)-inducible promoter. E. coli Rosetta2(DE3)pLysS cells harboring the expression vectors were cultured in LB liquid medium at 28 °C and IPTG was added when the culture reached 0.3 of an OD 600 . After 3 h induction, cells were harvested by centrifugation and suspended in 1 × PBS buffer (containing 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 PO 4 , and 1.8 mM KH 2 PO 4 ). Cells were disrupted with a sonicator (Astrason XL2020; Misonix, NY, USA). The cell debris was removed by centrifugation, and the supernatant was transferred to a 5 mL GSTrap HP column (GE Healthcare) on an ÄKTA explorer 10S (GE Healthcare). The N-terminal GST tag was removed by PreScission protease (GE Healthcare) at 4 °C for 16 h on a GST affinity column. Proteins were eluted and dialyzed against 1 × PBS. The eluted recombinant proteins have an additional N-terminal GPLGS sequence prior to the translational initiation codon. After homogeneity was verified by SDS-PAGE with Coomassie or silver staining, the purified proteins were divided into small volumes, and stored at 4 °C or − 20 °C until further use. Absorption spectra were recorded by a UV spectrometry on a U-2800A (Hitachi High-Tech Science Corp., Tokyo, Japan), Nano Drop 2000 (Thermo Fisher Scientific), or Cary 8454 UV-Vis Diode Array System (Agilent Technologies, CA, USA). Protein concentrations were determined by the Bradford method as described in the instructions accompanying the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA) with BSA (Thermo Fisher Scientific) as a standard.